Project Summary. Aging is a major risk factor associated with many leading causes of death in the United States, including type II diabetes, cancer, and neurodegenerative diseases. Dietary restriction (DR), the reduction in nutrient intake without causing malnutrition, is increasingly recognized to improve health and longevity across species. Yet, a complete mechanistic understanding of how the pro-health benefits of DR are conferred is lacking. Closing this gap in knowledge is essential to developing interventions which harness DR- related pathways for the benefit of treating aging-related diseases and improving human health. Work in Caenorhabditis elegans has shown DR improves longevity through sensory neuron-specific engagement of the evolutionarily conserved transformation growth factor-beta (TGF-β) signaling pathway. Other independent studies have implicated improved protein homeostasis (proteostasis) through signaling of the unfolded protein response of the endoplasmic reticulum (UPRER) as an essential contributor to DR- mediated lifespan extension. However, if and how UPRER and TGF-β signaling may converge to confer DR- mediated health improvements is unknown. The conserved C. elegans AMPylase, FIC-1 (FICD orthlog), helps maintain proteostasis in the ER via post- translational AMPylation of the BiP/Grp78 ortholog, HSP-3. BiP/Grp78 AMPylation inhibits its chaperone capacity and alters UPRER activation. We recently showed increased FIC-1 activity results in decreased TGF-β signaling from ASI sensory neurons in response to pathogen exposure; TGF-β signaling from ASI neurons is also required for DR-induced longevity extension. My preliminary data show loss of FIC-1 results in increased body size, suggestive of increased neuronal TGF-β signaling. Interestingly, FIC-1 loss increases longevity under DR compared to wild type, and this extension requires HSP-3. I also find acute starvation increases FIC- 1 expression in worm sensory neurons and increases HSP-3 AMPylation. Furthermore, we find that AMPylation of human BiP increases in response to nutrient deprivation in cellulo. Taken as a whole, these results are consistent with a model in which FICD/FIC-1-mediated BiP/HSP-3 AMPylation reduces the pro-longevity effects of DR by reducing TGF-β signaling. The proposed project will utilize novel C. elegans models to elucidate how ASI-specific AMPylation of HSP-3 may limit UPRER activity, TGF-β signaling, and DR- mediated benefits. It will also use established in cellulo models to investigate how starvation affects UPRER and TGF-β signaling in mammalian systems. The results of my work discern how UPRER and TGF-β signaling are intertwined in the context of DR, advancing knowledge in the aging and proteostasis fields. The long-term goal is to use this knowledge to develop interventions which decrease the deleterious impact of human aging.